Actin, villin, and calcium play key roles in regulating the finger-like shape of intestinal microvillar membranes. In physiologic calcium concentrations (less than MuM) villin crosslinks actin filaments into a bundle which stabilizes the microvillus membrane. However, when the calcium concentration increases greater than MuM, villin fragments the filaments into short pieces and the unsupported membrane vesiculates. Villin is unusual in that it possesses both actin filament bundling and fragmenting activities. In other cells these activities are properties of separate proteins. To understand how actin binding proteins regulate cell structure and motility we will identify the calcium-regulated changes in villin-actin binding domains using biochemical and structural methods. The goals of this proposal are to describe the molecular mechanisms of villin-mediated actin filament bundle formation and actin filament fragmentation. The specific aims are: 1. to map the positions of cysteine, methionine, and tryptophan residues in villin and actin with an antibody end-labeling method. 2. to identify and characterize actin-binding domains of villin using site specific chemical and proteolytic cleavage, binding assays and gel overlay methods. 3. to map the positions of villin-actin binding domains by chemical crosslinking and antibody end-labeling methods. 4. to identify and characterize the calcium binding sites on villin. 5. to start high resolution structural studies on villin by crystallizing villin fragments or villin fragments bound to actin. The molecular details of calcium-villin-actin interactions have important health-related significance because membrane vesiculation is an early event in the pathogenesis of intestinal malabsorption disorders such as coeliac syndrome and enterotoxin-induced enteropathy.